The proportion of Plasmodium vivax malaria among all malarias is increasing worldwide. Treatment with 8-aminoquinolines remain the only radical cure. However, 8-aminoquinolines can cause severe hemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient patients. The population of the multi-ethnic Chittagong Hill Tracts (CHT) carry the highest malaria burden within Bangladesh. As in many countries the national treatment guidelines recommend 8-aminoquinoline based radical cure without routine G6PD deficiency (G6PDd) testing to guide treatment. Aim of this study was to determine the need for routine testing within a multi-ethnic population by assessing the prevalence of G6PDd among the local population. Participants from 11 ethnicities were randomly selected and malaria status was assessed by microscopy, rapid diagnostic test (RDT) and polymerase chain reaction (PCR). G6PD status was determined by spectrophotometry and G6PD genotyping. The adjusted male median (AMM) was defined as 100% G6PD activity, participants were categorized as G6PD deficient (<30% activity), G6PD intermediate (30% to 70% activity) or G6PD normal (>70% activity). Median G6PD activities between ethnicities were compared and the association between G6PD activity and malaria status was assessed. 1002 participants were enrolled and tested for malaria. G6PD activity was measured by spectrophotometry in 999 participants and host G6PD genotyping undertaken in 323 participants. Seven participants (0.7%) had peripheral parasitaemia detected by microscopy or RDT and 42 by PCR (4.2%). Among 106 participants (32.8%) with confirmed genotype, 99 (93.4%) had the Mahidol variant. The AMM was 7.03U/gHb with 90 (9.0%) G6PD deficient participants and 133 (13.3%) with intermediate G6PD activity. Median G6PD activity differed significantly between ethnicities (p<0.001), proportions of G6PD deficient individuals ranged from 2% to 26% but did not differ between participants with and without malaria. The high G6PDd prevalence and significant variation between ethnicities suggest routine G6PDd testing to guide 8-aminoquinoline based radical in the CHT and comparable settings.